HSL3 Regulates Stomatal Closure And The Drought Stress Response By Modulating Hydrogen Peroxide Homeostasis

Drought stress imposes a major restriction on crop productivity all over the world.One of the most important part of the plant response to this stress is the control imposed on stomatal closure.In Arabidopsis thaliana,stomatal closure is effected by a change in the volume of the pair of guard cells which surround each stomata.This stomatal movement is mediated by the phytohormone ABA,which accumulates in response to drought stress.ABA exerts this effect through its activation of anion channel currents and inhibition of inward K+ channel currents.A common event associated with the response of plants to a diversity of abiotic stresses(including drought stress)is the accumulation of reactive oxygen species(ROS)such as O₂-,H₂O₂ and HO-.High concentrations of ROS damage plant cells,whereas at low concentrations,ROS function as signaling molecules,controlling plant growth and the stress response.In particular,the guard cells use H₂O₂ as a signaling molecule within the ABA signaling pathway.During ABA-mediated stomatal closure,H₂O₂ acts as a secondary messenger to regulate transmembrane ion trafficking.As the molecular mechanism of controling over the level of H₂O₂ in ABA pathway remains largely unknown.Here,our study was focused on the LRR-RLK protein HSL3,which has been shown to have the ability to negatively regulate stomatal closure by modulating the level of H₂O₂ in the guard cells.HSL3 was also markedly up-regulated by treating plants with either ABA or H₂O₂,as well as by dehydration.In our study,we first conducted experiments and analysis combined with genetic analysis on the tissue expression pattern and inducer of HSL3,so as to clarify the specific expression of HSL3 in Arabidopsis thaliana leaves and the fact that HSL3 can be induced by ABA and its downstream second messenger H₂O₂.Then,we conducted the physiological characters experiment of mutant,overexpression and so on from both macroscopic and microscopic dimensions.In order to further verify whether the rich leucine receptor kinase HSL3 affects stomatal closure and drought stress response by regulating intracellular homeostasis of hydrogen peroxide,we prove that the expression of HSL3 quantity will affect the defend the intracellular accumulation of ROS(especially the accumulation of H₂O₂),hydrogen peroxide generated and remove the expression of related genes and catalase enzyme activity from different angle of qualitative or quantitative experiments.Finally,we verified the conclusion from both positive and negative directions with different blocking agents with pharmacological methods,and confirmed that HSL3 plays an important role in ABA pathway by regulating hydrogen peroxide homeostasis.Guard cells shrink in response to drought stress and abscisic acid(ABA)signaling,thereby reducing stomatal aperture.Hydrogen peroxide(H₂O₂)is an important signaling molecule acting to induce stomatal closure.In the loss-of-function hsl3 mutant,both stomatal closure and the activation of slow-type anion currents proved to be hypersensitive to ABA treatment,and the mutant was more tolerant than the wild type to moisture deficit;the over-expression of HSL3 had the opposite effect.In the hsl3 mutant,the transcription of genes encoding proteins involved in both H₂O₂ production and H₂O₂ scavenging in the leaf,as well as the level of catalase activity was only weakly inducible by ABA,allowing H₂O₂ to accumulate in the guard cells.HSL3 was concluded to participate in the regulation of the response to moisture deficit through ABA-induced stomatal closure triggered by the accumulation of H₂O₂ in the guard cells.HSL3 can modulate hydrogen peroxide homeostasis,as hydrogen peroxide is not stable at room temperature and can slowly decompose itself into water and oxygen spontaneously.So the content of hydrogen peroxide in cells is maintained in a state of dynamic equilibrium for a long time.The production and clearance of hydrogen peroxide in cells are regulated by many genes and result in a complex network.NOX enzyme on the plasma membrane is a key enzyme to produce hydrogen peroxide in the ABA signaling pathway.It can catalyze NAPDH and oxygen and produce superoxide ions,which are then oxidized to hydrogen peroxide by superoxide dismutase(SOD).AtRbohD and AtRbohF are the two catalytic subunits encoding the enzyme NOX in Arabidopsis thaliana.When these two genes are inhibited,ABA stimulation will not increase the content of hydrogen peroxide and the stomata will not be closed.In mutants unable to produce the enzyme NADPH oxidase,ABA-promoted H₂O₂ production and stomatal closure are both impaired,resulting in a hyper-sensitivity to drought stress.The ROS scavenging enzymes catalase,ascorbate peroxidase and glutathione peroxidase have evolved as a major mechanism preventing the over-accumulation of H₂O₂.In rice,the protein DST regulates stomatal movement by controlling the synthesis of a precursor of the peroxidase used to neutralize H₂O₂ in the guard cells,while it has emerged that H₂O₂ can also be scavenged by glutathione peroxidase.The identity of the genes regulating the expression of H₂O₂ scavenging enzymes in the guard cells remains largely unknown.In our study,HSL3 has been proved to have the ability to adjust stomatal aperture by regulating the H₂O₂ level in guard cells,and as a result,affects the drought tolerance of plants.